Use of a strain of the bacillus siamensisspecies as a biosolution in mushroom cultivation

Abstract

The present invention relates to the use of a particular bacterial species as a biosolution in the production and cultivation of mushrooms. More particularly, the invention relates to the use of a Bacillus siamensis strain, deposited with the CNCM under the accession number CNCM I-5921, in the stimulation of the development and growth of mycelia or mushrooms or the improvement of the yields of edible mushroom crops, and in the control of the main fungal pathogens of edible mushroom crops.

Description

[0001] USE OF A STRAIN OF THE SPECIES BACILLUS SIAMENSIS AS A BIOSOLUTION IN FUNGUS CULTIVATION

[0002] The present invention relates to the use of a particular bacterial species as a biosolution in the production and cultivation of mushrooms. More specifically, the invention relates to the use of a strain of Bacillus siamensis, deposited with the National Collection of Microorganism Cultures (CNCM) under the order number CNCM 1-5921, in stimulating the development and growth of a mycelium or a fungus or improving the yields of edible mushroom crops, and in controlling the main fungal pathogens of edible mushroom crops.

[0003] Edible mushrooms have been used by humans since time immemorial, particularly for their nutritional value and medicinal properties. Many varieties of mushrooms exist, but the most widely cultivated worldwide are the button mushroom (Agaricus bisporus), oyster mushrooms (Pleurotus ostreatus, Pleurotus eryngii, etc.), and shiitake (Lentinula edodes). The button mushroom alone accounts for over 40% of the global market, oyster mushrooms 25%, and shiitake 15%. The main players in the global market are China, which produces by far the largest share of the world's edible mushrooms, the United States, and Europe.

[0004] On an industrial scale, the mushroom production method can be broken down into three stages: the first involves obtaining mycelium inoculum, the second involves producing fruiting substrates, and the third involves the actual cultivation of the mushrooms, using the mycelia and substrates obtained in the first two stages. These three stages can be carried out independently by different parties—the first by a mycelium producer, the second by a substrate producer, and the third by a mushroom grower—or by a single party, the mushroom grower. Obtaining mycelium inoculum begins with the inoculation of a sterilized culture medium using spores or a piece of inoculum.The culture medium can be agar, such as potato dextrose agar (PDA), a liquid medium such as potato dextrose broth (PDB), or any other nutrient solution, with or without a gelling agent. The development and growth of the mycelium on its culture medium are variable but generally range from 7 to 28 days, depending on the fungal strain. Once the mycelium has fully colonized the culture medium, a portion of the medium is then removed for inoculation with a colonization substrate. A suitable colonization substrate can be synthetic or, more commonly, composed of grains, typically rye, millet, sorghum, wheat, barley, rice, or oats, which have been previously sterilized and packaged in jars or micro-perforated culture bags.

[0005] The mycelium inoculum thus obtained is then used in the cultivation and production of mushrooms. The inoculum is therefore used to inoculate a fruiting substrate.

[0006] In the case of the button mushroom, the fruiting substrate is a compost generally composed of straw and animal manure, watered thoroughly to ensure its maturation over two to three weeks. The fruiting substrate is then pasteurized for a few days with decreasing temperatures from 60 to 40°C. Inoculation of the fruiting substrate takes place after pasteurization, for example, using a inoculating machine, by mixing the inoculum contained in its colonization substrate with the fruiting substrate. This is followed by an incubation period during which the inoculated fruiting substrates are placed in an enclosed room where the temperature, humidity, and oxygen are controlled for two weeks. The temperature is maintained at 22 to 25°C. The next step is casing, which consists of covering the fruiting substrate with a layer of suitable soil.The casing soil is, for example, a mixture of crushed and disinfected tuffeau limestone and horticultural peat. After a controlled drop in temperature, the first mushroom heads emerge from the fruiting substrate, and harvesting can then begin. The harvesting of button mushrooms follows several fruiting cycles called flushes, or alternating periods of harvesting and dormancy. The first two flushes are by far the most productive, with 50% of the total harvest attributed to the first flush and 35% to the second. The harvest cycle repeats approximately weekly. Harvesting can continue until the third flush, but yields decline rapidly.

[0007] In the current trend, mushroom growers are seeking to maximize their yields by producing in only two batches for reasons of productivity but also hygiene, because the 3 èmeTheft is often a gateway to the development of diseases and their longer-term establishment.

[0008] This last point is particularly important since the mushroom industry, currently undergoing significant transformation, is seeking effective and environmentally friendly bio-solutions that can help it navigate the ongoing agroecological transition more smoothly. Any product that increases yields of the first harvests while preserving production quality allows for both shorter production cycles and energy savings, as well as preventing the spread of diseases that represent a growing threat.

[0009] Mushroom cultivation is known to be one of the most sustainable in the world. Its production methods utilize the unparalleled biodegradation capabilities of the fungi kingdom to produce healthy food from agricultural waste and composted mixtures of plant and animal matter. When considered per unit area, mushroom cultivation remains one of the healthiest and most nutritious, while also being energy and water efficient, degrading or improving a wide variety of organic materials.

[0010] The case of urban mushroom farms is particularly striking since they exclusively consume food waste or unsold goods produced in their immediate surroundings. The number of these urban farms is increasing rapidly because they focus on the production of exotic species such as oyster mushrooms (Pleurotus ostreatus, Pleurotus eryngii, etc.) and shiitake (Lentinula edodes), known for being easier to cultivate.

[0011] Another great advantage of mushroom cultivation is the value of the compost produced at the end of the harvest, regardless of the mushroom species. Called champost or SMS (Spent Mushroom Substrate), it is highly sought after by farmers as a soil amendment for field crops because it is very balanced and rich in nutrients and biodiversity. The main fungal pathogens affecting mushroom cultivation worldwide are Dactylium dendroides (also known as Cladobotryum spp.), Verticillium fungicola (also known as Lecanicillium fungicola), and Mycogone perniciosa (also known as Hypomyces perniciosus), which can appear as early as the first days of harvest, attacking the primordia and fruiting bodies, rendering them unfit for consumption. Competitive fungi, such as Trichoderma aggressivum (also known as Trichoderma spp.), are also present.colonize the fruiting substrate and compete directly with the cultivated fungus, drastically reducing the amount of available nutrients.

[0012] These specialized and extremely virulent pathogens spread all the more rapidly when mushroom cultivation takes place in enclosed environments, under shelters and in temperature and humidity conditions particularly conducive to their development.

[0013] Yield losses can range from 20% to 100% in the worst-case scenario, and the economic impact on mushroom growers is even greater if the disease appears early, as there is no curative treatment. Only preventative measures can limit the emergence of these pathogens, such as applying plant protection products as soon as cultivation begins, or disinfecting equipment and fruiting rooms using chemical disinfectants or energy-intensive heat treatments.

[0014] Several effective plant protection products were still available to mushroom growers until recently, such as Sporgon® (prochloraz-Mn) and Banko® (chlorothalonil), which are now banned in Europe. Vivando® (metrafenone), the only one still authorized, now shows only reduced effectiveness.

[0015] For several years, all pathogens have gradually developed resistance to synthetic molecules, which, banned one after another, now leave them completely free to thrive. The result in Europe is alarming, with a growing number of mushroom growers left utterly helpless, witnessing diseases that had disappeared reappear and become permanently established.

[0016] In order to find ways to control fungal fruiting body diseases, several trials have been conducted in the past to test commercially available biocontrol products, such as Serenade® (Bacillus velezensis QST 713), Amylo-X® (Bacillus amyloliquefaciens D747), Serifel® (Bacillus amyloliquefaciens MBI 600), and Sonata® (Bacillus pumilus QST 2808). None of them demonstrated efficacy against the main pathogens affecting fungal crops, namely Dactylium dendroides, Verticillium humus fungicola, and Mycogonus perniciosa. Serenade® and Amylo-X® proved effective only against the so-called competitor fungus, Trichoderma aggressiveivum.

[0017] The mushroom industry is therefore in dire need of effective and environmentally friendly bio-solutions that increase crop yields from the very first harvests, shorten production cycles, and prevent diseases from developing and becoming entrenched. To be sustainable, the solutions developed must be safe for human health and remain easy to use, seamlessly integrating into the existing technical practices of mushroom cultivation.

[0018] The use of biostimulants in agricultural production methods is rapidly expanding. Biostimulants are substances capable of stimulating the metabolism of a plant, or fungus, and its natural nutrient uptake processes. More specifically, Regulation (EU) 2019 / 1009 of the European Parliament and of the Council of 5 June 2019, which entered into force on 22 July 2022, defines a biostimulant as "a product which stimulates plant nutrition processes independently of the nutrients it contains, with the aim of improving one or more of the following characteristics of plants or their rhizosphere: (a) nutrient use efficiency, (b) tolerance to abiotic stress, (c) qualitative characteristics, (d) the availability of nutrients confined in the soil or rhizosphere."

[0019] The aim of the present invention is to identify and propose an effective biostimulant and biocontrol agent suitable for mushroom production and cultivation methods. In particular, the biostimulant and biocontrol agent according to the invention should stimulate mycelium and fungal development and improve the yields of edible mushroom crops, or at the very least, prevent the inhibition of development and the resulting loss of these yields, and combat the main fungal pathogens affecting edible mushroom crops. Finally, the biostimulant and biocontrol agent according to the invention should be usable in conventional methods of substrate and mushroom production.

[0020] It is for this purpose that the applicant company conducted its research and discovered that a species of Bacillus exhibited surprising biostimulation capabilities on the development and yields of edible mushroom crops, and in the fight against the main fungal pathogens of edible mushroom crops, particularly when added to their culture media or substrates, and could therefore be used in mushroom cultivation processes and methods.

[0021] For this purpose, the invention relates to an isolated strain of the species Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921.

[0022] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of the bacterial strain isolated from Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921, for the stimulation of the development and growth of a mycelium or a fungus, or the improvement of the yields of fungal cultures.

[0023] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of the bacterial strain isolated from Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921, to combat the development of pathogens in the cultures of a fungus such as Dactylium dendroides, also known as Cladobotryum spp., Verticillium fungicola, also known as Lecanicillium fungicola, Mycogone perniciosa, also known as Hypomyces perniciosus, and Trichoderma aggressivum, also known as Trichoderma spp.

[0024] The bacterial strain isolated in France by the filing company was identified as Bacillus siamensis according to the NCBI (National Center for Biotechnology Information) database, after genotypic identification by Sanger sequencing.

[0025] This Gram-positive bacterium is aerobic and facultatively anaerobic, and capable of producing ellipsoidal endospores. It is highly motile with peritrichous flagella surrounding the cell. This species is distinguished by its ability to divide over a wide temperature range, from 4°C to 55°C, with an optimal growth temperature of 37°C. It forms creamy-white, mucous, semi-opaque, semi-domed colonies that grow 3 to 4 mm in diameter around the point of inoculation after 2 to 3 days of incubation at 25°C on PDA.

[0026] The Bacillus genus is prized in biotechnology and very present in microbial fermentation on an industrial scale because its robustness and rapid growth in bioreactors allow it to withstand aggressive processes such as spray drying, which is much less expensive than freeze-drying to obtain a powder of dehydrated cells, particularly stable over time.

[0027] The depositing company deposited the bacterial strain at the CNCM (National Collection of Microorganism Cultures) of the Pasteur Institute, which validated it and assigned it the order number CNCM 1-5921.

[0028] In button mushroom cultivation, the CNCM 1-5921 strain was shown to significantly improve yields, reaching +21% at the end of cultivation, compared to the untreated control, after a single spraying on the casing soil at a concentration of 1.10 10 cells / m 2, without any significant adverse effect on crop quality.

[0029] In parallel, the CNCM 1-5921 strain has been shown to metabolize octenol (CAS No. 3391-86-4) in in vitro tests. This volatile organic compound (VOC), also known as mushroom alcohol, is produced by the growing mycelium and tends to inhibit or even block fruiting initiation and primordia formation when present in excessive quantities. Octenol, on the other hand, has a biostimulating effect on the growth and development of the CNCM 1-5921 bacterial strain. This particularly important and unique discovery may explain why the CNCM 1-5921 strain significantly increases mushroom yields after being sprayed on the surface of the crops. Conversely, ethylene (CAS No. 74-85-1), a plant hormone also produced by the growing mycelium, can inhibit its own development if the concentration of this compound is too high.Some bacteria, such as the CNCM strain 1-5921, can produce ACC-deaminase, an enzyme that degrades a precursor in ethylene synthesis and thus accelerates mycelial growth.

[0030] In nature, there exist bacteria known as PGPB (Plant-Growth-Promoting Bacteria) or FGPB (Fungal-GPB), and even, exceptionally, MGPB (Mycelium-GPB and / or Mushroom-GPB). The CNCM 1-5921 bacterial strain from the applicant company is found to be both PGPB and MGPB, through biological mechanisms, some of which have been elucidated by internal research. This dual capability is all the more interesting because the strain according to the invention can also improve the yields of an agricultural crop amended with compost from the end of mushroom cultivation to which it had previously been applied.

[0031] After application of the fungal pathogen Dactylium dendroides, which is particularly problematic due to its virulence and persistence in mushroom farms (causing up to 40% yield losses), the CNCM 1-5921 strain maintains yields significantly higher than the untreated control. Although less effective than Sporgon®, a prochloraz-Mn-based pesticide now banned in Europe, the CNCM 1-5921 strain allows, in a single application at 2.10 9 cells / m 2 to maintain statistically comparable yields until the end of 2 ème stolen, up to 76% of the effectiveness achieved by Sporgon®. A more concentrated application at 5.10 9 cells / m 2 This allows us to raise this comparative efficiency to 81% at the end of 2 èmestolen, and at 65% at the end of the growing season. At the same time, this single application significantly reduces disease pressure by 90%, compared to Sporgon® at the end of 2 ème stolen, and 64% at the end of cultivation with a 2 ème This discovery is particularly important and unique because it demonstrates that the CNCM 1-5921 strain also has curative potential on an already infected mushroom crop when applied late. Indeed, no biocontrol agent, nor even any plant protection product, was able to contain fungal pathogens already present at the time of its application to mushroom crops. The re-emergence of diseases is now a major threat to mushroom growers who no longer have any effective means of control at their disposal, hence the need for them to have rapid access to long-term solutions.

[0032] Following application in fungal crops of the fungal pathogen Verticillium fungicola, which causes 20% yield losses worldwide, a single spraying of strain CNCM 1-5921 at a concentration between 2.10 5 and 2.10 9 cells / m 2 , helps to lower the disease pressure after the 2 ème fly (-32%), until the end of the growing season (-32%). The CNCM 1-5921 strain also helps maintain higher yields of healthy mushrooms while reducing the mass of diseased mushrooms, which contribute to spreading infectious outbreaks within crops.

[0033] In direct in vitro confrontation, the bacterial strain CNCM 1-5921 proves particularly effective against the main pathogens of Dactylium dendroides, Verticillium fungicola, Mycogone perniciosa, and Trichoderma aggressivum fungal cultures. The invention provides a sustainable and environmentally friendly solution for mushroom growers, eliminating the need for chemical fungicides and equipment sterilization, a technique that is effective in the short term but particularly energy-intensive.

[0034] The use of biostimulants and broad-spectrum biocontrol agents, naturally present in the environment, is a true innovation for mushroom production. The use of products with biostimulant effects has a major impact on mushroom production. Indeed, the main limitation for mushroom producers lies in their ability to quickly produce, store, and distribute their mushrooms to their customers. The use of biostimulants and biocontrol agents according to the invention allows producers to shorten their production cycles by increasing yields from the first harvests and preventing the onset and establishment of diseases. The addition of biostimulants and biocontrol agents can thus be carried out during the different stages of compost maturation or during the preparation of the casing soil, particularly after the casing soil has been applied to the compost.Thus, during trials conducted on casing soil, crops sprayed with the Bacillus siamensis species selected according to the invention showed a significant increase in crop yield, an increase in the number of mushrooms produced, and, in general, an improvement in the quality of the harvested mushrooms, even in the presence of fungal pathogens. The Bacillus strain selected according to the invention also proved capable of delaying and limiting the development of fungal pathogens.

[0035] The Bacillus siamensis species selected within the framework of the invention has the particularity of exhibiting a strong biostimulant effect, effective over a wide range of concentrations when applied by watering or spraying, adapted to the technical routes of mushroom growers.

[0036] Thus, the present invention relates to the use of a bacterial strain of Bacillus siamensis, or of the CNCM 1-5921 strain, for the improvement of mushroom yields under healthy culture conditions, and a reduction in the number of infectious foci and an increase in harvests of healthy mushrooms in the presence of fungal pathogens of the crops.

[0037] Furthermore, during mushroom cultivation, the introduction of the CNCM 1-5921 strain according to the invention increases yields from the first flushes and maintains yields in the presence of pathogens, while limiting their spread. The effects on the mushroom industry are therefore accompanied by economic and practical benefits for producers at each stage:

[0038] Energy savings linked to the possibility of shortening production cycles by reducing the duration of mushroom cultivation while maintaining adaptable yields for mushroom growers; raw material savings through better utilization of fruiting substrates linked to increased mushroom yields and improved sanitary quality; savings on synthetic plant protection products and reduction of residues in food for consumers and soil pollution from the addition of contaminated mushroom substrate.

[0039] Energy savings through the possibility of limiting particularly energy-intensive and costly heat treatments, thanks to the reduction of diseases; spray application easily adaptable to all production methods and combinable with the use of other biostimulants and supplements (nutritional supplements).

[0040] Application of a sustainable and environmentally friendly bio-solution to the cultivation of the main mushrooms grown worldwide.

[0041] The bacterial species Bacillus siamensis formulated within the framework of the invention can be used simply and effectively in conventional methods of mushroom production, without the need to complicate these methods.

[0042] Advantageously, the bacterial strain is in a solid form such as a powder or granules, or in a liquid form, preferably aqueous.

[0043] A product containing the bacterial strain according to the invention can thus be in liquid form, such as a concentrated suspension (CS), or in solid form, such as a dry powder obtained by fermentation in a bioreactor followed by spray-drying or by the addition of a cryoprotectant before freeze-drying. The dry powder can be in the form of a wettable powder (WP or WDP = Water Dispersible Powder) or soluble granules (WG or WDG = Water Dispersible Granules). The Bacillus siamensis species can optionally be produced in one of the forms described above and then encapsulated.

[0044] As an example, soluble powder containing dehydrated bacterial cells is prepared as follows:

[0045] Release of the bacterial strain from cryopreservation,

[0046] Subculturing the strain onto PDA culture media,

[0047] Strain growth in an incubator at 37°C,

[0048] Nutrient solution preparation in a 5-liter bioreactor,

[0049] Centrifugation of the solution after a 48-hour cycle,

[0050] Retrieval and washing of the pellet,

[0051] Spraying of the incoming liquid into the atomization chamber, drying under reduced pressure.

[0052] Obtaining a dry powder,

[0053] Packaging.

[0054] Preferably, the mycelium or fungus is chosen from among the basidiomycetes, in particular the button mushroom, oyster mushrooms and shiitake, more specifically Agaricus bisporus, Pleurotus ostreatus, Pleurotus eryngii and Lentinula edodes.

[0055] The invention further relates to a method of cultivating mushrooms comprising the following steps:

[0056] (a) preparation of a fruiting substrate,

[0057] (b) inoculation of the fruiting substrate with a mycelium inoculum,

[0058] (c) incubation,

[0059] (d) possibly, preparation of casing clay,

[0060] (e) possibly, gobetage,

[0061] (f) possibly, scratching,

[0062] (g) fruiting,

[0063] (h) harvests,

[0064] (i) end of harvest, the method being characterized in that a bacterial strain of Bacillus siamensis, or the bacterial strain isolated from the species Bacillus siamensis deposited with the CNCM under CNCM order number 1-5921, or a composition comprising therein, is applied at least once during at least one of the steps (a) to (i).

[0065] Preferably, according to this method, the bacterial strain is applied in the form of a liquid solution.

[0066] Preferably, the bacterial strain is applied by watering or spraying. Preferably, the bacterial strain is applied within the substrate or to the surface of the fungal cultures so that the concentration is between 10 3 cells / m 2 and 10 12 cells / m 2 or between 10 3 cells and 10 12 cells for 30L of substrate.

[0067] The invention further relates to the use of the compost from the end of the cultivation of a mushroom obtained by the method described above as an amendment or fertilizer for an agricultural crop.

[0068] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of an isolated strain of Bacillus deposited with the CNCM under the order number CNCM 1-5921, to metabolize, or biodegrade, the octenol produced by a mycelium or a fungus.

[0069] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of an isolated strain of Bacillus deposited with the CNCM under the order number CNCM 1-5921, to prevent the synthesis of ethylene produced by a mycelium or by a fungus.

[0070] The features of the invention mentioned above, as well as others, will become clearer upon reading the following description of exemplary embodiments.

[0071] Example 1: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the yields of Agaricus bisporus crops

[0072] 1.1 Preparation of a solution comprising the strain according to the invention.

[0073] A solution is formulated based on the following elements:

[0074] Microorganism: Bacillus siamensis bacterial strain deposited with the CNCM under order number CNCM 1-5921 in the form of a dry powder, obtained by spray drying.

[0075] The dry powder is obtained as follows: multiplication of the bacterial strain by fermentation, drying by spray drying at the end of fermentation, packaging.

[0076] The resulting dry powder is intended to be dissolved in water before application. This will be referred to hereafter as "CNCM 1-5921 solution".

[0077] 1.2 Experimental protocol for application on casing soil

[0078] Application of the CNCM 1-5921 solution in the mushroom production phase

[0079] Inoculation of 9 kg plots of compost (each representing 0.1 m²) 2 mushroom culture) with 72 g of untreated mycelium.

[0080] Incubation of the plots for 13 days.

[0081] Deposit of 3000 mL of casing soil (90% black peat and 10% calcium carbonate) at a thickness of 3 cm, on each plot.

[0082] Scratching the casing soil, 7 days after casing.

[0083] Spraying the casing soil, 7 days after casing, with CNCM 1-5921 solution (as described in point 1.1), so as to obtain a concentration of 2.10 9 5.10 9 and 1.10 10 cells / m 2 , solubilized in 150 mL of water per plot.

[0084] Post-incubation of the plots for 10 days.

[0085] Beginning of the induction of fruiting of the first flight.

[0086] Harvesting, counting the number of mushrooms, weighing and measuring the yields of the 3 flights.

[0087] Each treatment described above was conducted on 6 identical experimental plots, thus corresponding to 6 replicates. The mycelium strain used for all experimental trials was the commercial variety of Agaricus bisporus, brand name Delta, produced by the American company Amycel. 1.3 Results

[0088] Table 1 below presents the measured yields, in kilograms of healthy mushrooms per square meter of culture, of the different experimental treatments described above. All yields are also expressed as a percentage, relative to the control treatment.

[0089] Table 1]

[0090] Newman-Keuls test (5% threshold)

[0091] Table 2 below shows the number of mushrooms counted, in units per square meter of culture, for each experimental treatment described above. The number of mushrooms harvested is also expressed as a percentage, relative to the control treatment.

[0092] Ta b.2]

[0093] Newman-Keuls test (5% threshold) According to the results of the experimental tests of Example 1, a clear improvement in yields can be observed when the CNCM 1-5921 solution is applied to the casing soil.

[0094] Increasing the doses applied by spraying resulted in a statistically significant improvement in mushroom yields (Newman-Keuls test at the 5% risk threshold), with a 21% increase at the end of the growing season at a concentration of 1.1 O 10 cells / m 2 .

[0095] The number of mushrooms harvested also increases with the action of the CNCM 1-5921 solution, as the application dose increases.

[0096] The CNCM 1-5921 solution therefore allows mushroom growers to optimize the use of raw materials in the composition of their growing substrates, but also to save energy.

[0097] The CNCM 1-5921 solution also ensures sufficient yields from the first harvests to shorten their production cycle and avoid the appearance of diseases which would involve the use of energy-intensive methods or polluting and currently ineffective plant protection products.

[0098] In conclusion, the CNCM 1-5921 solution, applied by simple spraying, increases mushroom crop yields from the very first harvests. Its biological action optimizes the use of raw materials and saves energy during cultivation, while also preventing the spread of diseases for which mushroom growers no longer have lasting solutions.

[0099] Example 2: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the crop yields of Agaricus bisporus in the presence of the pathogen Dactylium dendroides

[0100] 2.1 Experimental protocol for application on casing soil

[0101] Application of the CNCM 1-5921 solution in the mushroom production phase

[0102] Inoculation of 9 kg plots of compost (each representing 0.1 m²) 2mushroom culture) with 72 g of untreated mycelium. Incubation of the plots for 13 days.

[0103] Deposit of 3000 mL of casing soil (90% black peat and 10% calcium carbonate) at a thickness of 3 cm, on each plot.

[0104] Scratching the casing soil, 7 days after casing.

[0105] Spraying the casing soil, 7 days after casing, with CNCM 1-5921 solution (as described in point 1.1), so as to obtain a concentration of 2.10 9 cells / m 2 , solubilized in 150 mL of water per plot.

[0106] Spray the casing soil 9 days after casing with the fungicide Vivando® to obtain a concentration of 1 mL / m². 2 , solubilized in 150 mL of water per plot.

[0107] Spraying onto the casing soil, 11 days after casing, of a solution containing spores of the pathogen Dactylium dendroides, so as to obtain a concentration of 2.10 6 spores / m 2 , solubilized in 150 mL of water per plot.

[0108] Post-incubation of the plots for 10 days.

[0109] Beginning of the induction of fruiting of the first flight.

[0110] Harvesting, counting the number of infected foci, weighing and measuring the yields of the 3 flights.

[0111] Each treatment described above was conducted on 6 identical experimental plots, thus corresponding to 6 replicates. The mycelium strain used for all experimental trials was the commercial variety of Agaricus bisporus, brand name Delta, produced by the American company Amycel. 2.2 Results

[0112] Table 3 below presents the measured yields, in kilograms of healthy mushrooms per square meter of culture, of the different experimental treatments described above. All yields are also expressed as percentages, relative to the control treatment.

[0113] Table 3]

[0114] Table 4 below shows the number of webs that appeared, or foci of the pathogen Dactylium dendroides, in units per square meter of culture, for each experimental treatment described previously. The number of webs that appeared is also expressed as a percentage, relative to the control treatment.

[0115] Table 4]

[0116] According to the results of the experimental tests of Example 2, it can be observed that the application of the CNCM 1-5921 solution makes it possible to maintain yields of healthy mushrooms, superior to the fungicide Vivando® at the end of the culture.

[0117] A single spray of CNCM 1-5921 solution at a low concentration of 2.10 9 cells / m 2 This strain reduces disease pressure after the first two flushes (-13%) and throughout the growing season (-9%). It should be noted that Vivando®, a metrafenone-based fungicide, showed no efficacy in this experimental trial. In conclusion, the strain according to the invention, in the presence of the pathogen Dactylium dendroides, maintains higher yields of healthy mushrooms while reducing the number of outbreaks of infection, which are particularly feared by mushroom growers due to the lack of effective solutions.

[0118] Example 3: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the yields of Agaricus bisporus crops in the presence of the pathogen Dactylium dendroides

[0119] 3.1 Experimental protocol for application on casing soil of mushrooms

[0120] Inoculation of 9 kg plots of compost (each representing 0.1 m²) 2 mushroom culture) with 72 g of untreated mycelium.

[0121] Incubation of the plots for 13 days.

[0122] Deposit of 3000 mL of casing soil (90% black peat and 10% calcium carbonate) at a thickness of 3 cm, on each plot.

[0123] Scratching the casing soil, 7 days after casing.

[0124] Spraying the casing soil, 7 days after casing, with CNCM 1-5921 solution (as described in point 1.1), so as to obtain a concentration of 2.10 9 cells / m 2 , solubilized in 150 mL of water per plot.

[0125] Spraying the casing soil, 9 days after casing, with the fungicide Sporgon®, to obtain a concentration of 1 g / m² 2 , solubilized in 150 mL of water per plot.

[0126] Spraying onto the casing soil, 11 days after casing, of a solution containing spores of the pathogen Dactylium dendroides, so as to obtain a concentration of 5.10 6 spores / m 2 , solubilized in 150 mL of water per plot.

[0127] Post-incubation of the plots for 10 days.

[0128] Start of the induction of fruiting of the first flush. Spraying of the casing soil, 1 to 3 additional times depending on the method (as detailed in point 3.2), with CNCM 1-5921 solution, so as to obtain a concentration of 2.10 9 cells / m 2 , solubilized in 150 mL of water per plot and per application.

[0129] Harvesting, counting the number of infected foci, weighing and measuring the yields of the 3 flights.

[0130] Each treatment described above was conducted on 6 identical experimental plots, thus corresponding to 6 replicates. The mycelium strain used for all experimental trials was the commercial variety of Agaricus bisporus, brand name Delta, produced by the American company Amycel.

[0131] 3.2 Results

[0132] Table 5 below presents the measured yields, in kilograms of healthy mushrooms per square meter of culture, of the different experimental treatments described above. All yields are also expressed as percentages, relative to the control treatment.

[0133] Table 5]

[0134] Newman-Keuls Test (5% Threshold) Table 6 below presents the number of webs that appeared, or foci of the pathogen Dactylium dendroides, in units per square meter of culture, for each experimental treatment described previously. The number of webs that appeared is also expressed as a percentage, relative to the control treatment.

[0135] Ta b.6]

[0136] According to the results of the experimental trials in Example 3, it can be observed that an application of the CNCM 1-5921 solution maintains yields of healthy mushrooms, statistically comparable to the Sporgon® fungicide at the end of 2 ème flight (Newman-Keuls test at the 5% risk threshold). A single spraying ensures 77% of the yield maintenance achieved by applying Sporgon® after 2 flights, and 45% at the end of the growing season.

[0137] A single spray of CNCM 1-5921 solution at a low concentration of 2.109 cells / m 2 This helps reduce disease pressure after the first two harvests (-50%). It should be noted that Sporgon®, a fungicide based on prochloraz-Mn, has not been authorized in the European Union since 2021.

[0138] In conclusion, the strain according to the invention, in the presence of the pathogen Dactylium dendroides, allows for the maintenance of higher yields of healthy mushrooms while reducing the number of outbreaks of infection, which are particularly feared by mushroom growers in the absence of effective solutions. Example 4: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the yields of Agaricus bisporus crops in the presence of the pathogen Dactylium dendroides

[0139] 4.1 Experimental protocol for application on casing soil

[0140] Application of the CNCM 1-5921 solution in the mushroom production phase

[0141] Inoculation of 9 kg plots of compost (each representing 0.1 m²) 2 mushroom culture) with 72 g of untreated mycelium.

[0142] Incubation of the plots for 13 days.

[0143] Deposit of 3000 mL of casing soil (90% black peat and 10% calcium carbonate) at a thickness of 3 cm, on each plot.

[0144] Scratching the casing soil, 7 days after casing.

[0145] Spraying the casing soil, 7 days after casing, with CNCM 1-5921 solution (as described in point 1.1), so as to obtain a concentration of 5.10 9 and 1.10 10 cells / m 2 , solubilized in 150 mL of water per plot.

[0146] Spraying the casing soil, 9 days after casing, with the fungicide Sporgon®, to obtain a concentration of 1 g / m² 2 , solubilized in 150 mL of water per plot.

[0147] Spraying onto the casing soil, 11 days after casing, of a solution containing spores of the pathogen Dactylium dendroides, so as to obtain a concentration of 4.10 6 spores / m 2 , solubilized in 150 mL of water per plot.

[0148] Post-incubation of the plots for 10 days.

[0149] Beginning of the induction of fruiting of the first flight.

[0150] Spraying onto the casing soil, at the end of the ère stolen (as detailed in point 4.2), from the CNCM 1-5921 solution, so as to obtain a concentration of 5.10 9 and 1.10 10 cells / m 2 , solubilized in 150 mL of water per plot.

[0151] Harvesting, counting the number of infected foci, weighing, and measuring the yields of the three flights. Each treatment described above was carried out on six identical experimental plots, thus corresponding to six replicates. The mycelium strain used for all experimental trials was the commercial variety of Agaricus bisporus, brand name Delta, produced by the American company Amycel.

[0152] 4.2 Results

[0153] Table 7 below presents the measured yields, in kilograms of healthy mushrooms per square meter of culture, of the different experimental treatments described above. All yields are also expressed as percentages, relative to the control treatment.

[0154] Ta b.7]

[0155] Table 8 below shows the number of webs that appeared, or foci of the pathogen Dactylium dendroides, in units per square meter of culture, for each experimental treatment described previously. The numbers of webs that appeared are also expressed as percentages, relative to the control treatment. [Table 8]

[0156] Newman-Keuls test (5% threshold)

[0157] According to the results of the experimental tests in Example 4, it can be observed that an application of the CNCM 1-5921 solution maintains yields of healthy mushrooms, comparable to the Sporgon® fungicide at the end of 2 ème stolen. A single spray at 5.10 9 cells / m 2 It allows us to ensure 81% of the yield maintenance allowed by the application of Sporgon® after 2 flights, and 65% at the end of the crop.

[0158] At the same time, this single application at 5.10 9 cells / m 2enabled a significant reduction in disease pressure of 90%, compared to Sporgon® at the end of 2 ème flight (Newman-Keuls test at the 5% risk threshold), and 64% at the end of culture after a 2 ème application. This discovery is particularly important and unique because it demonstrates that the CNCM 1-5921 solution also has curative potential on an already infected fungal culture, which is unprecedented. Indeed, the 2 ème late application to 5.10 9 cells / m 2 was carried out after the l ère stolen, in the presence of several infectious foci, testifying to the early onset and virulence of a fungal pathogen that was already well established.

[0159] In conclusion, the strain according to the invention, in the presence of the pathogen Dactylium dendroides, maintains higher yields of healthy mushrooms while significantly reducing the number of outbreaks of infection, including in post-infection curative applications. Example 5: Evaluation of the impact of the bacterial strain CNCM 1-5921 applied according to the invention on the yields of Agaricus bisporus crops in the presence of the pathogen Verticillium fungicola

[0160] 5.1 Experimental protocol for application on casing soil

[0161] Application of the CNCM 1-5921 solution in the mushroom production phase

[0162] Inoculation of 9 kg plots of compost (each representing 0.1 m²) 2 mushroom culture) with 72 g of untreated mycelium.

[0163] Incubation of the plots for 13 days.

[0164] Deposit of 3000 mL of casing soil (90% black peat and 10% calcium carbonate) at a thickness of 3 cm, on each plot.

[0165] Scratching the casing soil, 7 days after casing.

[0166] Spraying the casing soil, 7 days after casing, with CNCM 1-5921 solution (as described in point 1.1), so as to obtain a concentration of 2.10 5 and 2.10 9 cells / m 2 , solubilized in 150 mL of water per plot.

[0167] Spraying the casing soil, 9 days after casing, with the fungicide Sporgon®, to obtain a concentration of 1 g / m² 2 , solubilized in 150 mL of water per plot.

[0168] Spraying onto the casing soil, 11 days after casing, of a solution containing spores of the pathogen Verticillium fungicola, so as to obtain a concentration of 2.10 6 spores / m 2, solubilized in 150 mL of water per plot.

[0169] Post-incubation of the plots for 10 days.

[0170] Beginning of the induction of fruiting of the first flight.

[0171] Harvesting, weighing, and measurement of healthy and diseased mushroom yields from the three flights. Each treatment described above was conducted on six identical experimental plots, thus corresponding to six replicates. The mycelium strain used for all experimental trials was the commercial variety of Agaricus bisporus, brand name Magnum, produced by the American company Amycel.

[0172] 5.2 Results

[0173] Table 9 below presents the measured yields, in kilograms of healthy mushrooms per square meter of culture, of the different experimental treatments described above. All yields are also expressed as percentages, relative to the control treatment.

[0174] Ta b.9]

[0175] Table 10 below presents the measured yields, in kilograms of diseased fungi per square meter of culture, of the different experimental treatments described above. All yields are also expressed as a percentage, relative to the control treatment.

[0176] Ta b.10] According to the results of the experimental tests of Example 5, it can be observed that an application of the CNCM 1-5921 solution makes it possible to maintain yields of healthy mushrooms superior to the untreated control, throughout the mushroom culture.

[0177] A single spray of CNCM 1-5921 solution at a low concentration of 2.10 5 at 2.10 9 cells / m 2This product helps reduce disease pressure after the first two flushes (-32%) until the end of the growing season (-32%). It should be noted that Sporgon®, a fungicide based on prochloraz-Mn, has not been authorized in the European Union since 2021.

[0178] In conclusion, the strain according to the invention makes it possible, in the presence of the pathogen Verticillium fungicola, to maintain higher yields of healthy fungi while reducing the mass of diseased fungi, which contribute to spreading infectious foci within crops.

[0179] Example 6: Evaluation of the impact of the bacterial strain CNCM 1-5921 on the growth and development of the pathogens Dactylium dendroides, Verticillium fungicola, Mycogone perniciosa and Trichoderma aggressivum.

[0180] 6.1 Experimental protocol under laboratory conditions

[0181] In vitro confrontation of the bacterial strain CNCM 1-5921 with fungal cultures

[0182] Preparation of PDA (Potato Dextrose Agar) poured into Petri dishes.

[0183] Incubate the Petri dishes at 23°C for 7 days.

[0184] Longitudinal deposition of the bacterial strain CNCM 1-5921 with a loop.

[0185] Deposition of an inoculum of the pathogens Dactylium dendroides, Verticillium fungicola, Mycogone perniciosa and Trichoderma aggressivum.

[0186] Incubation of inoculated Petri dishes at 23°C for 14 days.

[0187] Observation and photography of the front and back of Petri dishes. 6.2 Results

[0188] Figures 1 to 4 illustrate the impact of the bacterial strain CNCM 1-5921 on the growth and development of the pathogens Dactylium dendroides (Fig.1), Verticillium fungicola (Fig.2), Mycogone perniciosa (Fig.3) and Trichoderma aggressivum (Fig.4), after 14 days of incubation.

[0189] According to the results of the experimental tests of Example 6, it can be observed that the bacterial strain CNCM 1-5921 produces a particularly inhibitory effect on the growth and development of the pathogens Dactylium dendroides, Verticillium fungicola, Mycogone perniciosa and Trichoderma aggressivum after 14 days of incubation.

[0190] It is noteworthy that this effect is long-lasting, not just for a few days, and that it occurs remotely, without any direct contact with the bacterial strain CNCM 1-5921 being necessary, except for Trichoderma aggressivum.

[0191] In conclusion, the bacterial strain CNCM 1-5921 proves particularly effective against the main pathogens of mushroom cultures, namely Dactylium dendroides, Verticillium fungicola, Mycogone perniciosa, and Trichoderma aggressivum. The strain according to the invention thus provides mushroom growers with a sustainable and environmentally friendly solution, eliminating the need for chemical fungicides and the sterilization of equipment and infrastructure—a technique that is effective in the short term but particularly energy-intensive.

[0192] Example 7: Evaluation of the impact of octenol, or mushroom alcohol, on the bacterial strain CNCM 1-5921.

[0193] 7.1 Experimental protocol under laboratory conditions

[0194] In vitro confrontation of the bacterial strain CNCM 1-5921 with octenol

[0195] Preparation of PDA (Potato Dextrose Agar) poured into Petri dishes.

[0196] Incubate the Petri dishes at 23°C for 7 days. Deposit and spread 0.5 pL, 1 pL, 2 pL and 4 pL of octenol (CAS No. 3391-86-4) with a rake.

[0197] Longitudinal deposition of the bacterial strain CNCM 1-5921 with a loop.

[0198] Incubation of inoculated Petri dishes at 23°C for 14 days.

[0199] Observation and taking photos of Petri dishes.

[0200] 7.2 Results

[0201] Figure 5 illustrates the impact of octenol, or mushroom alcohol, on the bacterial strain CNCM 1-5921, after 14 days of incubation.

[0202] According to the results of the experimental tests of Example 7, it can be observed that octenol produces a biostimulating effect on the growth and development of the bacterial strain CNCM 1-5921. It should also be noted that this effect increases as the volume of octenol applied increases, from the untreated control (a), to 0.5pL (b), 1pL (c), 2pL (d) and 4pL (e).

[0203] This discovery is particularly important because octenol is produced by the growing mycelium and tends to inhibit or even block fruiting initiation and primordia formation when present in excessive quantities. Dispersing excess octenol on the surface of the cultures requires the use of fans, which consume energy and are not always effective.

[0204] In conclusion, octenol biostimulates the growth and development of the CNCM 1-5921 bacterial strain, which is clearly capable of degrading or metabolizing it. This aspect is particularly interesting and unique, as it may explain why the CNCM 1-5921 solution significantly increases the yields of healthy fungi (Example 1), even in the presence of disease.

Claims

DEMANDS 1) Isolated strain of the species Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921. 2) Use of the bacterial strain according to claim 1, for the stimulation of the development and growth of a mycelium or a fungus or the improvement of the yields of fungal cultures. 3) Use of the bacterial strain according to claim 1, to control the development of pathogens in the cultures of a fungus such as Dactylium dendroides, also known as Cladobotryum spp., Verticillium fungicola, also known as Lecanicillium fungicola, Mycogone perniciosa, also known as Hypomyces perniciosus, and Trichoderma aggressivum, also known as Trichoderma spp. 4) Use according to any one of claims 2 to 3, wherein the bacterial strain is in a solid form such as a powder or granules, or in a liquid form, preferably aqueous. 5) Use according to any one of claims 2 to 4, wherein the mycelium or fungus is selected from basidiomycetes, in particular button mushroom, oyster mushrooms and shiitake, more particularly Agaricus bisporus, Pleurotus ostreatus, Pleurotus eryngii and Lentinula edodes. 6) Method for culturing fungi from a mycelium inoculum, comprising the following steps: (a) preparation of a fruiting substrate, (b) inoculation of the fruiting substrate with a mycelium inoculum, (c) incubation, (d) possibly, preparation of casing clay, (e) possibly, gobetage, (f) possibly, scratching, (g) fruiting, (h) harvests, (i) end of harvest, characterized in that the bacterial strain according to claim 1, or a composition comprising it, is applied at least once during at least one of the steps (a) to (i). 7) Method according to claim 6, wherein the bacterial strain is applied in the form of a liquid solution. 8) A method according to any one of claims 6 to 7, wherein the bacterial strain is applied in the mass of the substrate or on the surface of fungal cultures so that the concentration is between 10 3 cells / m 2 and 10 12 cells / m 2 or between 10 3 cells and 10 12 cells for 30L of substrate. 9) Method according to any one of claims 6 to 8, wherein the bacterial strain is applied by watering or spraying. 10) Use of a final culture compost of a mushroom obtained by a method according to claim 6 as an amendment or fertilizer for an agricultural crop. 11) Use of a bacterial strain according to claim 1, to metabolize or biodegrade octenol produced by a mycelium or by a fungus. 12) Use of a bacterial strain according to claim 1, to prevent the synthesis of ethylene produced by a mycelium or by a fungus.

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